Conjugate comprising an active agent, a polypeptide and a polyether

ABSTRACT

The present invention relates to conjugates, comprising an active substance, a polypeptide and a polyether, a process for the production of such conjugates as well as their use.

This is a national phase filing of the Application No. PCT/DE96/02487,which was filed with the Patent Corporation Treaty on Dec. 20, 1996, andis entitled to priority of the German Patent Application 195 48 114.3,filed Dec. 21, 1995.

1. FIELD OF THE INVENTION

The present invention relates to conjugates, comprising an activesubstance, a polypeptide and a polyether, a process for the preparationof such conjugates as well as their use.

2. BACKGROUND OF THE INVENTION

There is a great demand of concentrating active substances at their siteof action. This applies particularly to active substances for treatingtumors and inflamed tissues, respectively. It is very often tried toobtain the active substance concentration by administration of highamounts of active substance. However, this leads to considerableside-effect in patients, particularly in their livers and kidneys.

Therefore, it is the object of the present invention to provide apossibility of concentrating active substances at their site of action.

3. SUMMARY OF THE INVENTION

The present invention relates to conjugates, comprising an activesubstance, a polypeptide and a polyether, a process for the preparationof such conjugates as well as their use.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a conjugate according to the invention, comprisingpolylysine (PL), 4 polyethylene glycols (PEG), 8 aminofluoresceins (AF1)and 2 diethylenetriaminepentaacetates (DTPA) labeled with ¹¹¹In, AF1being linked via cyanuric chloride to free amino groups of PL, and DTPAbeing linked to PL via an acid amide bridge (SEQ ID NO:1).

FIG. 2 shows the distribution of the conjugate of FIG. 1 according tothe invention, measured by means of scintiscanning, in tumor tissue.

5. DETAILED DESCRIPTION OF THE INVENTION

It is the object of the present invention to provide a possibility ofconcentrating active substances at their site of action. According tothe invention this is achieve by the subject matters defined in theclaims.

Thus, the subject matter of the present invention relates to a conjugatecomprising an active substance, a polypeptide and a polyether.

The expression “active substance” comprises substances of any kind whichcan be used for treating and/or diagnosing a disease, particularly atumoral disease and/or an inflammatory disease. Examples of suchsubstances are chemotherapeutic agents, photoactive substances,radioactive substances, fluorescence-capable substances or substancescapable of emitting fluorescence, substances for fluoro-nuclear magneticresonance spectroscopy, substances as radiosensitizers and/or substanceshaving binding sites for metallic compounds. Examples of thechemotherapeutic agents are virostatics, antiprotozoals, cytostaticagents and antibiotics. Representatives thereof are e.g. doxorubicin,daunorubicin, tetracyclines, antimetabolites such as methotrexate, andderivatives thereof. Examples of photoactive substances are porphyrins,such as o-, m- and/or p-tetracarboxyphenylporphyrin (TCPP), chlorins andbacteriochlorins. An example of the radioactive substance is a tyrosinelabeled with radioactive iodine. Examples of the substances capable ofemitting fluorescence are fluorescent dyes, such as fluorescein,aminofluorescein (AF1) as well as derivatives and analogues thereof.Examples of the substance for fluoro-nuclear magnetic resonancespectroscopy are polyfluorocarboxylic acids such as trifluoroaceticacid. An example of the radiosensitizer substance is m-aminobenzoicamide. Examples of the substances having a binding site for metalliccompounds are substances which include hydroxyl, carbonyl or carboxylgroups. They may have one or more binding sites, preferably at least 2,more preferably 3 to 6. If several binding sites are present, they maybe the same or differ from one another. Examples of the substanceshaving binding sites for metallic compounds areethylenediaminetetraacetate, diethylenetriaminepentaacetate (DTPA),triethylenetetraaminehexaacetate, alizarin and derivatives thereof.Substances having binding sites for metallic compounds favorably have adetectable metallic compound which may contain or consist of one or moredetectable metals and/or metal ions. Examples of such metals are Zn, Cu,Co, Fe, Ni, Pt, Gd and In, which are preferably bivalent or trivalent,Gd³ ⁻ being especially preferred. The metals and/or metal ions may beradioactive, such as ¹¹¹In.

One or more of the above active substances may be present in theconjugate according to the invention. If several are present, they maybe the same or differ from one another.

The expression “polypeptide” comprises polypeptides of any kind whichhave up to 50 amino acids, preferably 7 to 30 amino acids, especiallypreferably 10 to 30 amino acids. The polypeptide may have equal ordifferent amino acids. The amino acids are preferably those havingamino, carboxyl or phenolic OH-groups in the side groups. They may benon-naturally occurring and/or naturally occurring amino acids such aslysine, tyrosine, asparaginic acid and glutamic acid. Especiallypreferred polypeptides are polylysine (PL)1, polytyrosine,polyasparaginic acid and polyglutamic acid.

A person skilled in the art is familiar with the above polypeptides.They can be purchased or be prepared by known techniques, e.g. theMerriefield technique.

A polyether is present in the conjugate according to the invention. Itprotects the conjugate particularly from excretion via the kidneys andprovides it with a good water solubility. The polyether preferably has amolecular weight of 2,000 to 5,000 daltons. The polyether may beetherified or esterified at the terminal hydroxyl group by a C₁-C₁₂alkyl group, particularly methyl group. The polyether is preferably apolyethylene glycol (PEG), such as methoxypolyethylene glycol, and isbound to the polypeptide via active groups such as methoxypolyethyleneglycol-p-nitrophenylcarbonate, methoxypolyethylene glycolsuccinimidylsuccinate, methoxypolyethylene glycol tresylate,methoxypolyoxyethyleneamine, methoxypolyoxyethylene-carboxylic acid andmethoxypolyoxyethyleneimidazolcarbonyl.

The conjugate according to the invention may also contain several of theabove polyethers, preferably 2 to 6. They may be the same or differ. Ifseveral polyethers are present, the sum of the molecular weights thereofis favorably 10,000 to 50,000 daltons.

In the conjugate according to the invention, the individual componentscan be linked directly, e.g. covalently, via an acid amide or acid esterbond, or indirectly via a linker such as cyanuric chloride. Both theactive substance and the polyether are linked preferably to thepolypeptide.

The above components of the conjugate according to the invention aregiven as educts. Therefore, they are present in derivatized form in theconjugate according to the invention.

Examples of conjugates according to the invention are indicated in FIG.1 and Example 1.

A process for the preparation of an above conjugate is also providedaccording to the invention. In such a process, the polypeptide, theactive substance and the polyether are linked with one another as usual,preferably covalently. Reference is made to the preparation of aconjugate according to the invention in Example 1.

Conjugates according to the invention distinguish themselves byconcentrating well in tumors and tissues damaged by inflammatorydiseases. In addition, they can be charged with the most differingactive substances, particularly at the same time, without beginimmunogenic. This is a great advantage over prior art conjugates whichoften, when several active substances are simultaneously present, causeundesired immune responses in patients. Thus, the conjugates accordingto the invention are well suite for treating diseases, particularlytumoral and inflammatory diseases.

Furthermore, the conjugates according to the invention are well suitedfor the diagnosis of diseases, particularly tumoral and inflammatorydiseases. For example, the active substance of conjugates according tothe invention may be a substance capable of emitting fluorescence. Theseconjugates are given to the tumor patient e.g. before an operation. Theconjugates according to the invention then concentrate in the tumor.During the operation, the substance capable of emitting fluorescence canbe stimulated by light, e.g. a UV lamp, to emit fluorescence. Thus,healthy tissue can be distinguished from tumor tissue.

The below examples explain the invention in more detail. The followingpreparations and examples are given to enable those skilled in the artto more clearly understand and to practice the present invention. Thepresent invention, however, is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and methods which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

6. EXAMPLES a. Example 1 Preparation of a Conjugate According to theInvention, Comprising PL, 8 AF1, 2 DTPA and 4 MethoxypolyethyleneGlycols (MPEG)

50 mg of 5([4,6-dichlorotriazine-2-yl]amino) fluorescein (AF1, MW 531.7;Sigma) were dissolved in 4 ml of dimethylsulfoxide (DMS) and added to100 mg of polylysine (PL-MW˜1000 D, Sigma), dissolved in 2 ml DMSO and 2ml of distilled water. Then 1 ml 0.34 M NaHCO₃ was added, and theslightly turbid solution was allowed to stand or about 30 min.

1 g of methoxypolyethylene glycol (MPEG)—nitrophenylcarbonate wasdissolved in 10 ml of dioxan and added to the above AF1-PL solution. 50μl of 1 N NaOH were added thereto. The conversion was terminated afterabout 48 hours. PL-AFL-MPEG₄ was obtained. Unwanted accompanyingsubstances were separated by ultrafiltration (YM 10). The molecularweight was determined by means of GPC as usual.

PL-AF1-MPEG₄ was reacted with diethylenetriaminepentaacetic acid (DTPA)which was labeled with ¹¹¹In for the nuclear-medical use or withgadolinium (Gd) as tumor-penetrating MR contrast agent, as follows:

Diethylenetriaminepentaacetic acid (DTPA) was dissolved by heating to aconcentration of 20 mg/ml in DMSO. After cooling the clear solution toroom temperature, 1.5 to 2 time the molar amount ofN,N′-dicyclohexylcarbodiimide (DCC) and 5 times the molar amount ofN-hydroxysuccinimide (HS1) were added. After a reaction time of about 14to 15 h, the formation of the succinimidyl ester (DTPA-SE) wascompleted. It was used for the subsequent linkage to PL-AF1-MPEG₄.

For this purpose, the above solution of DTPA-SE was added very slowlyand with constant stirring to the above PL-AFL-MPEG₄ solution purifiedby ultrafiltration, the initially clear solution becoming turbid byexcess and unreacted DCC and still dissolved di-cyclohexylurea (DCCH)which are both insoluble in aqueous solution. Having terminated theaddition of DTPA-SE, the reaction mixture was stirred for full reactionat room temperature for another 60 minutes. Thereafter, the turbidmatter was separated via a sterile filter unit (Millipore, Stericup-CV,0.22 μm Low Binding Duropore Membrane) and the low-molecularwater-soluble components (DMSO, HSI and unbound DTPA) were separated byultrafiltration via a membrane with 10 kD exclusion limit (Amicon).Analytical purity was controlled by means of HPLC. ThePL-AFL-MPEG₄-DTPA₂ (A) conjugate according to the invention wasobtained.

For labeling (A) with ¹¹¹In, 10 to 20 μl of a 0.2 M Na-citrate solutionwere added to 185 Mbq ¹¹¹In (5 mCi) charged as 0.1 M of hydrochloricacid solution of ¹¹¹InCl₃ having a low carrier content. For thispurpose, about 5 mg (A), dissolved in 1 to 2 ml 0.17 M NaHCO₃ wereadded. Thereafter, the reaction mixture was separated by ultrafiltrationfrom unbound ¹¹¹In and citrate. For this purpose, the reaction volumewas filled up to 2 ml and inserted in a disposable ultrafiltration unit(Centricon C 30, Amicon), the separation of the low-molecularcontaminations taking place by centrifugation in a rotor which can beinclined by 45°. (A) labeled with ¹¹¹In was obtained.

The charging of (A) with Gd was carried out in a manner the same as thelabeling with ¹¹¹In, only greater substances amounts being required.

b. Example 2 Absorption of a Conjugate According to the Invention inTumor Tissue

a) Rats having a Walker 256 carcinosarcoma were given intravenouslyabout 70 μCi (2 mg/kg) of the conjugate of FIG. 1 according to theinvention. The distribution of the conjugate in the rats was shown byscintiscanning after 5 minutes, 1, 3, 24 and 48 hours. The result isshown FIG. 2.

FIG. 2 discloses that the conjugate according to the inventionconcentrates in the tumor but not in the cardiac or liver region.

b) The above rats were killed after 48 hours, and the radioactivitydistribution in the organs was determined. The results are shown inTable 1.

Table 1 discloses that the conjugate according to the inventionconcentrates in the tumor.

TABLE 1 Distribution of the conjugate of FIG. 1 according to theinvention in the blood, in certain organs and in the tumor of a rat. 30%of the radioactivity left in the body is found in the tumor which onlyconstitutes 4.8% of the body weight. amount of Moiety Weight/volumeConjugate (μCi) (%) rat 243.0 27.5  100% blood 15.4 ml 1.32 4.8% tumor11.2 g 8.50 30.9% skin 40.2 g 0.75 2.9% spleen 0.55 g 0.23 0.8% kidneys(together) 2.38 g 2.60 9.4% liver 10.71 g 2.82 10.2% gastrointestinaltract 13.30 g 1.43 0.5%

All references cited within the body of the instant specification arehereby incorporated by reference in their entirety.

What is claimed is:
 1. A conjugate for diagnosing or treating a tumor oran inflammatory disease in a subject comprising an active substance, apolypeptide and one to six polyethers, wherein each polyether isattached to the polypeptide via a side chain of the polypeptide, and theconjugate concentrates in the subject in the tumor or in a tissuedamaged by the inflammatory disease.
 2. The conjugate of claim 1,wherein the active substance is a substance useful for diagnosing atumoral disease in a subject, and the conjugate concentrates in a tumor.3. The conjugate of claim 1, wherein the active substance is achemotherapeutic agent, a photoactive substance, a radioactivesubstance, a substance capable of emitting fluorescence, a substance forfluoro-nuclear magnetic resonance spectroscopy, a substance asradiosensitizer or a substance having a binding site for metalliccompounds.
 4. The conjugate of claim 1, comprising several activesubstances.
 5. The conjugate of claim 1, wherein the polypeptideconsists of about 7 to about 30 amino acids.
 6. The conjugate of claim1, wherein the polypeptide comprises amino acids which have amino,carboxyl or phenolic OH groups in the side chains.
 7. The conjugate ofclaim 1, wherein the polypeptide consists of naturally occurring aminoacids.
 8. The conjugate of claim 7, wherein the amino acids are selectedfrom the group consisting of lysine, tyrosine, asparaginic acid andglutamic acid.
 9. The conjugate of claim 8, wherein the polypeptide ispolylysine, polytyrosine, polyasparaginic acid or polyglutamic acid. 10.The conjugate of claim 1, wherein the polyether has a molecular weightof 2,000 to 5,000 daltons.
 11. The conjugate of claim 1, wherein thepolyether is a polyethylene glycol.
 12. A process for the preparation ofa conjugate of claim 1, wherein the active substance, the polypeptideand the polyether are bonded covalently with one another.
 13. A methodfor treating a tumoral disease or an inflammatory disease, comprisingadministering the conjugate of claim
 1. 14. A method for diagnosing atumoral disease or an inflammatory disease, comprising administering theconjugate of claim
 1. 15. The conjugate of claim 1, wherein the activesubstance is a substance useful for treating a tumoral disease in asubject, and the conjugate concentrates in a tumor.
 16. The conjugate ofclaim 1, wherein the active substance is a substance useful fordiagnosing an inflammatory disease in a subject, and the conjugateconcentrates in a tissue damaged by an inflammatory disease.
 17. Theconjugate of claim 1, wherein the active substance is a substance usefulfor treating an inflammatory disease, and the conjugate concentrates ina tissue damaged by an inflammatory disease.